Seed transmission of Acidovorax avenae subsp. citrulli
is a key factor in the dissemination of bacterial fruit blotch of cucurbits. In
this study, we report on the survival and seed transmission of A. avenae subsp.
citrulli from watermelon (Citrullus lanatus) and melon (Cucumis
melo) seed lots that were more than 30 years old. The longest
previously-reported survival of A. avenae subsp. citrulli was from
19-year-old watermelon seed (4) and the longest reported survival for any
seedborne bacterial plant pathogen was 24 years for Curtobacterium
flaccumfaciens pv. flaccumfaciens from common bean seed (2).

Seedling grow-outs were conducted on 500 melon seed lots and 125 watermelon
seed lots from collections in Ames, IA, and Griffin, GA. All seed lots were
produced in the United States between 20 and 40 years ago and were conserved in cold
storage at 5°C or below. A few lots were known to be infected by A. avenae
subsp. citrulli, but little information was available for the majority.
The grow-out assay was based on one recognized as the USDA National Seed Health
System standard (www.seedhealth.org).
Fifty to 70 seeds from each lot were planted into 20 × 30 × 6-cm
bleach-sanitized plastic trays filled with soilless potting mix (SunGro
Horticulture, Bellevue, WA). Each tray was enclosed in a clear plastic bag which
raised the humidity to near 100% and prevented cross-contamination between
trays. Greenhouse temperatures were maintained at 24 to 28°C. Germinated
seedlings were observed through the plastic and the bags were only opened if
symptoms of water-soaking or necrosis were observed on the cotyledons (Fig. 1).
Bags were opened in isolation and latex gloves were used to collect samples for
preliminary testing by ELISA immunostrips (Agdia Inc., Elkhart, IN). Isolations
were made from immunostrip-positive plants by dilution plating onto King’s B
agar, and suspect colonies were selected for further testing. Pathogenicity was
tested by toothpick inoculations of known susceptible varieties ‘Edisto’ melon
and ‘Crimson Sweet’ watermelon. Isolates were further confirmed as A. avenae
subsp. citrulli by conventional PCR (3).

Seed transmission was detected from 10 of the 500 melon seed lots, seven of
which were 35 to 40 years old. All positive seed lots were grown a second time
for confirmation. Seed transmission was detected from 10 of the 125 watermelon
seed lots, the oldest being 34 years old. Due to limited seed supplies, a second
grow-out was done with two watermelon lots and both were confirmed as positive.
Generally, one to two infected seedlings were observed per lot.

Attempts to isolate the pathogen by dilution plating from seed washes of a 37-year-old and a 38-year-old melon seed lot were unsuccessful, perhaps due to low
bacterial numbers, bacterial dormancy, or competition from saprophytes. Problems
with isolation from old seed lots have been noted previously (1). However, A.
avenae subsp. citrulli was detectable in the seed washes by
immunostrips and, when seeds were planted into a sterile potting mix, positive
plants (Fig. 2) were identified in each of the two seed lots. This demonstrated
that viable, pathogenic bacteria were indeed present and multiplied on the host
tissue during germination.

The fact that A. avenae subsp. citrulli can survive over
35 years on dried seed suggests that the pathogen is highly resistant to aging
and may survive as long as the seed is viable. Refrigerator or freezer storage
improves seed longevity and probably improves chances for pathogen survival as
well. While the pathogen may not survive as long under different storage
conditions, this study demonstrates that it does have the capacity to survive
for decades on seed.